The present invention relates to a vaccinal preparation comprising an antigen and a hydrophobized polysaccharide. More specifically, it relates to a vaccinal preparation comprising antigens such as tumor-associated antigens and virus antigens, together with a hydrophobized polysaccharide which acts as an adjuvant, wherein the antigens and the hydrophobized polysaccharide form a complex. The vaccinal preparation of the present invention has, in particular, activating and inducing effects on cytotoxic T cells (abbreviated as xe2x80x9cCTLxe2x80x9d hereinafter in the specification) that are specific for the antigen, and therefore, the preparation is extremely useful as a vaccine for preventive and/or therapeutic treatment of cancers, viral diseases and autoimmune diseases.
CTLs have actions of specifically recognizing cancer cells or virus-infected cells through their T cell receptors or other, and destroying or damaging the cells. Their roles are important as a bio-defense mechanism against cancers or viruses. Recently, it has been revealed that T cell receptors of CTL do not directly recognize the specific antigen expressed on the surface of cancer cells or virus-infected cells. Instead, they recognize complexes including an MHC class I antigen, which is expressed on the surface of antigen-presenting cells, per se, such as macrophages, dendritic cells, cancer cells or virus-infected cells, together with an oligopeptide derived from the specific antigen bound to the MHC class I antigen (xe2x80x9cCTL epitopexe2x80x9d of the specific antigen).
It is considered that the complex of an MHC class I antigen/an oligopeptide derived from a specific antigen is formed by a processing as explained below. Antigenic proteins are synthesized in cytoplasm, and then some of the proteins are degraded into oligopeptides by intracellular protease complexes (xe2x80x9cproteasomexe2x80x9d). Some of the oligopeptides (consisting of 9-10 amino acid residues) are then transported from cytoplasm into the endoplasmic reticulum membrane by transporter proteins (TAP: transporter in antigen processing) that are present in the endoplasmic reticulum membrane, and oligopeptides having high affinity for the MHC class I antigen are preferentially bind to the MHC class I antigen and presented on the surfaces of the cells.
It has been attempted to develop vaccines that artificially can activate the CTL (CTL-activating vaccines) in order to provide a method of preventive and/or therapeutic treatment of cancers, viral diseases or autoimmune diseases by eliminating cancer cells, virus-infected cells or autoantigen-reactive lymphocytes. To achieve the aforementioned purpose, it is necessary to perform immunization of a living body with cancer cells or virus-infected cells, per se, which expresses specific antigens, or alternatively, to introduce the specific antigens or the oligopeotides derived therefrom into the processing machinery for the antigen presenting cells so that a complex with the MHC class I antigen can be expressed.
In order to develop such xe2x80x9cCTL-activating vaccinesxe2x80x9d, some attempts were made which include, for example, 1) a method comprising the step of transforming a gene encoding a specific antigenic protein by means of a viral vector; 2) a method comprising the step of introducing a specific antigenic protein having an appropriate molecular weight and several CTL epitopes into cytoplasm by means of a certain technique, and 3) a method comprising the step of binding an oligopeptide consisting of 9-10 amino acid residues as a potential CTL epitope directly to an MHC class I antigen of an antigen presenting cell.
Among them, method 1) is a so-called gene therapy, whose efficacy and safety have not yet been established. Efficacy obtained by using method 3) have been shown in animal studies; however, practical problems may arise for applications to humans. Since almost every patient has different kind of MHC class I antigens, it should be necessary to cover the diversity of CTL epitopes corresponding to the variety of antigens. In other words, it is necessary to elucidate amino acid sequential motifs of oligopeptides having high affinity for each kind of the MHC class I antigens, and to provide customized oligopeptides as medicaments that correspond to each of the MHC class I antigen. Therefore, the development of vaccines based upon this class will be extremely difficult.
As to the approach according to method 2), some successful examples are known and its efficacy and safety seem satisfactory. Therefore, the method has been expected as a most successful means to develop the CTL activating vaccines applicable to variety of patients. However, when a specific antigenic protein, per se as a polypeptide, is administered to a living body to activate specific CTLs, the protein is usually administered as a mixture with an adjuvant. For example, ISCOM (Takahashi et al., Nature, 344, 873-875, 1990), QS-21 (Newman et al., J. Immunol., 148, 2357-2362, 1992), mannan-coated liposomes (WO92/4887), AF (Raychaudhuri et al., Proc. Natl. Acad. Sci. USA, 89, 8308-8312, 1992) or other have been reported as such adjuvant.
It is known that polysaccharide/cholesterol derivatives can be used as polysaccharide coatings for liposomes [Japanese Patent Unexamined Publication (KOKAI) No.(Sho)61-69801/1986], or coatings for lipid emulsions [Japanese Patent Unexamined Publication (KOKAI) No.(Sho)63-319046/1988]. As explained above, it is also known that tumor-associated antigens or virus antigen/liposome complexes, coated with polysaccharides containing mannose, have inducing activity of CTL (WO92/4887).
However, all of the aforementioned products should be manufactured by a coating process using a polysaccharide/cholesterol derivative after a liposome formation or a lipid emulsification of an antigenic protein (an antigen). As for a complex consisting solely of a polysaccharide/cholesterol derivative and an antigenic protein (an antigen), anything has not been reported as to what effects can be specifically obtained by the complex.
As mentioned above, in order to activate specific CTLs by administering a kind of antigenic protein as a polypeptide, to human, it is necessary to use a certain adjuvant. However, no adjuvant has been available which achieves efficient immunization and safety, and can be readily prepared.
The inventors of the present invention conducted various studies to discover a substance which can be used as a simpler and more efficient adjuvant. As a result, they found that hydrophobized polysaccharides composed of naturally occurring polysaccharides modified with alkyl groups or cholesterol groups can encapsulate, quite readily and efficiently, tumor-associated antigenic proteins or virus antigenic proteins to form complexes without any need of phospholipids. They also found that specific CTLs were activated in animals administered with the complex and bio-defense reactions were induced. The present invention was achieved on the basis of these findings.
The present invention thus provides vaccinal preparations comprising a hydrophobized polysaccharide and an antigen, and vaccinal preparations comprising a complex of a hydrophobized polysaccharide and an antigen. According to preferred embodiments of the present invention, there are provided the aforementioned vaccinal preparations which contain an antigen encapsulated in microparticles comprising aggregated hydrophobized polysaccharides; and the aforementioned vaccinal preparation comprising the hydrophobized polysaccharide as an adjuvant. Those vaccinal preparations can be used for, for example, anti-cancer and anti-viral or autoimmune disease therapies.
According to preferred embodiments of the present invention, there are provided the aforementioned vaccinal preparations wherein the hydrophobized polysaccharide is a polysaccharide modified with an alkyl group or a sterol residue; the aforementioned vaccinal preparations wherein the hydrophobized polysaccharide is characterized to contain a saccharide unit, at a ratio of one to five per 100 saccharide units that constitute the polysaccharide, whose primary hydroxyl group is a group represented by the following formula (I):
xe2x80x94Oxe2x80x94(CH2)mCONH(CH2)nNHxe2x80x94COxe2x80x94Oxe2x80x94Rxe2x80x83xe2x80x83(I)
wherein R represents an alkyl group or a sterol residue; m represents 0 or 1; and n represents an arbitrary positive integer; the aforementioned vaccinal preparations wherein the polysaccharide is pullulan or mannan; and the aforementioned vaccinal preparations wherein the sterol residue is cholesterol residue.
According to further preferred embodiments of the present invention, there are provided the aforementioned vaccinal preparations wherein the antigen is a protein which is presented as an oligopeptide by an MHC class I antigen and induces cytotoxic T-cells; the aforementioned vaccinal preparations wherein the antigen is ErbB-2 protein; and the aforementioned vaccinal preparations wherein the antigen is a tumor-associated antigen, a viral antigen, or an autoantigen-reactive T-cell receptor.
As another aspect of the present invention, there are provided methods for immunization which comprises the step of administering to a mammal the aforementioned vaccinal preparations; and the aforementioned methods which are used for inducing antigen-specific cytotoxic T-cells. As a further aspect of the present invention, adjuvants comprising a hydrophobized polysaccharide are provided. As a preferred embodiment of the aforementioned invention, the adjuvants are provided which are used for the formulation of a vaccinal preparation together with an antigen. There is also provided a use of a hydrophobized polysaccharide for the manufacture of the aforementioned vaccinal preparations.